Infertility affects one in ten couples, resulting in millions of couples struggling to become pregnant. Many of these couples are potential candidates for infertility treatment. Follicle stimulating hormone (FSH), either extracted from urine or produced recombinantly, is a parenterally-administered protein product used by specialists to increase fertility and has been so used clinically since the 1960's. For example, FSH is used for ovulation induction (OI) and for controlled ovarian hyperstimulation (COH). Whereas OI is directed at achieving a single follicle to ovulate, COH is directed at harvesting multiple oocytes for use in various in vitro assisted reproductive technologies (e.g., for in vitro fertilization). FSH is also used in gonadotropin replacement therapy in males.
The use of FSH is limited by its high cost, the need for extensive monitoring by specialist physicians, by lack of oral dosing or other noninvasive routes of administration, and the need for daily patient injections. Recombinant FSH suffers from a short half-life and correspondingly diminished biopotency, necessitating frequent administration and limited clinical usefulness. For example, recombinant human FSH (hFSH) must be administered as a daily intramuscular or subcutaneous injection, often for 8 to 12 days or more when used for ovulation induction. These regimens are associated with a number of side effects, including local irritation and discomfort, which result in poor compliance and a reduction in therapeutic efficacy. Thus, there exists a need for forms of FSH with increased half-life and bioavailability as compared to traditional forms of FSH therapy.
Follicle stimulating hormone (FSH) is found in nature as a non-covalently linked heterodimeric protein consisting of an alpha (α) subunit and a beta (β) subunit (Pierce J G and Parsons T F (1981) Ann Rev Biochem 50:465-95). Subunit assembly has been reported to be essential for bioactivity of FSH (Jia X C and Hseuh A J W (1986) Endocrinology 119:1570-7) as well as for the stability of the beta subunit (Keene et al. (1989) J Biol Chem 264:4769-75).
One approach to improve FSH therapy has been by increasing the glycosylation of the protein. Other approaches have included carboxy terminal portion (CTP) extended forms of FSH (see e.g., U.S. Pat. No. 5,338,835, and U.S. 2003/0211580 A1) or FSH mimetics (see e.g., U.S. Pat. No. 6,653,338) for the treatment of infertility. Early attempts, however, to improve half-life and bioactivity have yet to result in a therapeutically effective drug capable of providing advantages over existing therapies.
It has also been reported that a single-chain fusion of the α and β subunits of FSH (single-chain FSH) is fully active (Sugahara et al. (1996) J Biol Chem 271:10445-8). Single-chain FSH is reported to have an increased serum half-life when fused with the carboxyterminal peptide of human chorionic gonadotropin (hCG). (See Klein et al. (2003) Hum Reprod 18:50-6; Bouloux et al. (2001) Hum Reprod 16:1592-7; Duijkers et al. (2002) Hum Reprod 17:1987-93.)
The use of heterodimeric FSH and its formulation are fraught with stability and purification issues not present with single-chain FSH. While recombinant FSH is known (see e.g., U.S. Pat. No. 5,767,251), keeping the alpha and beta subunits associated in a way that results in a biologically active molecule useful for therapeutic purposes, and in particular with long-acting forms of heterodimeric FSH, remained a challenge until the present invention.
The creation of fusion proteins comprised of immunoglobulin constant regions linked to a protein of interest, or fragment thereof, has been described (see, e.g., U.S. Pat. Nos. 5,155,027, 5,428,130, 5,480,981, and 5,808,029). These molecules usually possess both the biological activity associated with the linked molecule of interest as well as the effector function, or some other desired characteristic, associated with the immunoglobulin constant region. Fusion proteins comprising an Fc portion of an immunoglobulin can bestow several desirable properties on a fusion protein including increased stability, increased serum half-life (see Capon et al. (1989) Nature 337:525) as well as binding to Fc receptors such as the neonatal Fc receptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,030,613, and 6,485,726).
FcRn is active in adult epithelial tissue and expressed in the lumen of the intestines, pulmonary airways, nasal surfaces, vaginal surfaces, colon and rectal surfaces (U.S. Pat. No. 6,485,726). Fusion proteins comprised of FcRn binding partners (e.g., IgG, Fc fragments) can be effectively shuttled across epithelial barriers by FcRn, thus providing a non-invasive means to systemically administer a desired therapeutic molecule. Additionally, fusion proteins comprising an FcRn binding partner are endocytosed and protected by cells expressing the FcRn. Instead of being marked for degradation, these fusion proteins are recycled out into circulation again, thus increasing the in vivo half-life of these proteins.
FSH has been conjugated to Fc as described in U.S. 2003/0235536 A1. However, therein is described single-chain FSH-Fc fusion protein for delivery to the central airways of non-human primates. Single-chain FSH-Fc fusions contain hFSHβα-Fc, in a single-chain with the beta and alpha subunits conjugated end-to-end and the two polypeptide chains of the fusion are identical (see FIG. 1c). As shown in FIG. 1c, single-chain FSH-Fc forms a homodimer.